Bacterial biofilms are a considerable public health threat because they cause chronic and hospital-acquired infections, as well as the persistent biofouling of medical implants, but are resistant to antibiotics. Biofilm regulation by NO has been observed broadly in bacteria, thus therapeutic interventions based on NO signaling could have a significant impact on public health. Bacterial NO signaling is poorly understood, however. For example, NO regulation of biofilm dispersal in P. aeruginosa, a principal pathogen in cystic fibrosis and hospital-acquired infections, is very well documented, but the NO sensor remains unknown. To bridge this knowledge gap, a long-term goal of the PI is to determine the mechanism of NO signaling in bacteria and to use this knowledge as a foundation for developing therapeutic strategies to disperse biofilms. The PI has shown that NO regulates biofilm formation in many bacteria, including P. aeruginosa, but P. aeruginosa lack a homolog of the H-NOX protein shown to mediate the response to NO in other species. Thus, P. aeruginosa must have an alternate NO sensor. The PI has discovered a novel family of hemoproteins named NosP. Based on strong preliminary data it is hypothesized that NosP is a NO sensor that regulates biofilm formation. The objective of the proposed work is to characterize P. aeruginosa NosP and determine the role of this protein in NO-mediated control of biofilm formation. This proposal is innovative because it forges new logical connections between NO sensing and biofilm formation, establishing a new paradigm for the role of NO in bacteria. This proposal is significant because elucidation of the basis for NO signaling in P. aeruginosa will open new therapeutic opportunities for controlling infection caused by this important human pathogen. The hypothesis will be tested by pursuing three specific aims: (1) to determine the phenotype of nosP; (2) to gain structural and functional insights into NosP; and (3) to delineate the signaling mechanism downstream of NosP. Under aim 1, P. aeruginosa biofilm, virulence, and antibiotic resistance will be quantified in the presence of varying amounts of NO, using wild-type and NosP deletion and expression strains. Under aim 2, the structure and spectroscopy of NosP will be described and it will be determined if NosP reversibly binds NO concentrations consistent with biofilm dispersal. Under aim 3, it will be determined if NosP regulates the activity of a certain kinase to mediate biofilm formation through HptB/sRNA signaling. The PI has significant experience with the proposed assays. Upon completion of these aims, NosP is expected to be established as an NO sensor that regulates biofilm formation in P. aeruginosa. This would be a fundamentally important discovery because it will define a new signaling pathway and novel antibiotic targets, for which there is a pressing need, especially in light of the increased antibiotic resistance typically seen in biofilming organisms. In addition to this positive impact on public health, this proposal will have an important positive impact on future research, because it is the starting point for deeper investigations into the role of NO in biofilm regulation and bacterial/host interaction.